Charactarizing loop & needle volumes

<p>Hi ,</p><p></p><p>I have replaced the loop in my Acquity UPLC system to 5µl loop .after that I performed startup system and by mistake did not change in charactarize</p><p></p><p>loop & needle configuration the loop volume to 5µl as it should be ,instead the wrong loop volume (10µl) was entered .</p><p></p><p>The system performed the startup process without any fail message .In addition, when the same test was performed from Sample manager-->charactarize</p><p></p><p>neele & loop volumes ,and again with the wrong loop volume configured (10µl) it showed the the test is Pass and measured volume is 7.0µl for the loop .</p><p></p><p>On the other hand when I changed the loop volume to 5µl which is the real loop volume it showed that the test is Pass and the measured volume is 7.0µl</p><p></p><p>for the loop .</p><p></p><p>After this long introduction my question is the following :does the system inject the correct volume when placing 5µl loop ,configuring 10µl by mistake</p><p></p><p>and injecting 4µl volume ?</p><p></p><p>Thanks .</p><p></p><p>M.K</p>


  • Dear M.K.

    We ask the user to define the loop for the system and then to characterize it. The loop is not tracked electronically (as the column or now the detector flow cell is), so the system based its assumption that the loop was correctly ID'd and no error would be generated. The programmed/set loop volume from a programming perspective relates to the maximum programmable/allowed volume. Therefore if a 5uL Loop is physically installed but the programmed loop is set 10uL this will allow the user to enter a number up to 10uL, however the loop will only except 5uL physically. And of course the purpose of entering the loop volume is for documentation and for system records.

    However, to be clear, the characterize test is designed to measure the loop volume and if it passes by definition the loop is suitable. The test is not designed to check the validity of the label or the user entered definition of the loop's nominal volume. To the system a loop is a loop, it does not discriminate (or check the label claim if you will) and a 10 uL loop with a 7 uL calculated volume is acceptable and credible as 5 uL loop with an 7 uL volume. What the test is designed to do is measure what the effective "actual" volume is. This is because, as you will see from threads just before this, nominal volumes of loops can be inaccurate, and which you yourself proved, you have a rather big 5 uL loop. As for the characterizing at 7uL, a 5uL loop does have plus/minus tolerance. The tolerance is built into the characterization, therefore if it does pass the characterization, the loop is acceptable. You will see the allowed values in the other threads for reference.

    In other words - 4 uL injection volume that you asked the system to make was valid. You will note the test produced the same result for the loop whether you designated the nominal volume as 5 uL or 10 uL What  users must avoid is NOT doing the characterization test. Also remember you can inject 4 uL out of several loops, 5 through 250 uL. In this specific case the system would have thrown an error if you tried to inject 8 uL since the loop cannot attain that value.

    Hope this helps,



  • Thank you Liz .


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  • "The tolerance is built into the characterization, therefore if it does pass the characterization, the loop is acceptable."

    Just in relation to this I was wondering if someone could tell me at what tolerance would sample loop characterisation produce a 'fail' result? I have seen sample loops pass specification at 200% nominal volume. In a previous post about this topic I received a document relating to tolerances of different size loops. In this it states a range of values low, nominal and high which I am guessing are manufacturers specification and have no relation to what Acquity system software deems acceptable limits as characteristed volumes for sample loop 'pass' outside of these ranges.

    Can someone clarify this issue, please?

  • Hello

    I checked with software engineerng and this is the hard coded for allowed values compred to nominal volumes:-

    Loop tolerance (since initial release v1.0)

    Loop Size (ul)

    Tolerance (%)

    <= 5


    <= 10


    > 10


    Thus for smaller loop up to 100% is allowed. However, this si becasue this is the best that th manufacturers can attain for specifications.

    Hope this helps,


  • Thank you for providing this information.

  • Jepoi

    Has anyone encountered the error message when characterizing the loop and needle volumes which says that the sample manager failed to calibrate the system volume.

    Prior to this we were getting an error message on a sequence saying "Sample pressure low: needle damaged or no wash solvent" Solvent is present in the sample syringe. And the needle looks fine. Any ideas?


  • I just got an error message this morning saying the needle calibration is out of range (14.4).  The needle was taken out yesterday and replaced because I thought that was possibly the reason I was getting the injection failure message (turns out the filter in the weak wash solution was out of the liquid so the needle didn't have to be changed).  Yesterday when I characterized the needle and loop volumes it was okay.  I couldn't find any troubleshooting information about what to do if the needle calibration is out of range so I did the static leak tests on the sample syringe, wash syringes and needle seal and the back pressure regulator test.  The sample syringe static leak test failed but the rest passed.  The following shows the results after the sample syringe test:

    Test: Failed

    Test pressure: 150 psi

    Compressed Volume: 7.6 uL

    Decompressed Volume: 6.6 uL

    Start pressure: 16 psi

    End pressure: 0 psi

    Pressure change: 0 psi

    Average pressure leak: 0 psi/sec

    Any suggestions about what else I can do for the needle calibration problem or how to correct the sample syringe leak test?  I've checked the fittings on the sample syringe and they seem tight.  I also don't see any bubbles in the sample syringe.

  • DavDag

    Starting with the original error reported "Sample pressure low: needle damaged or no wash solvent" Solvent is present in the sample syringe. This could simply be empty or not primes wash solvents. I would suggest to verify the wash solvent are full, the tube/filter is located at the bottom of the bottle and the wash solvent are primed adequately. There should not be any air bubbles in the wash syringes.

    As for the second error message "failed to calibrate the system volume". Again I would suggest to check the simple things first which would be to prime the sample syringe adequately and look for air bubbles in it. Additionally once that is done you should perform a VDD calibration 3 time consecutively all calibrations should be very close to each other.

  • Jepoi

    Thanks for the inputs. Apparently, it was a bad needle. I replaced the needle as soon as we received the new one and the issue was resolved.